Abstract Members of the genus Chlamydia are Gram-negative bacteria that cause various acute as well as chronic diseases in humans. Although chlamydiae share numerous aspects of their typical bacterial life style, such as intracellular developmental cycle and the preference to primarily invade epithelial cells, these human pathogens employ sophisticated species-specific strategies contributing to an extraordinary diversity in tissue tropism and disease manifestation. C. trachomatis infects mucosal surfaces of the human urogenital tract or columnar epithelial cells of the eye. Chlamydia-mediated genital infections represent the leading cause of bacterial STDs in the United States, with 1,708,569 infections reported to the CDC in 2017. C. pneumoniae infects mucosal surfaces of the human respiratory tract, causing pneumonia, bronchitis, pharyngitis, and sinusitis. Epidemiological data suggest that most people are infected and reinfected throughout life. Infections with both chlamydial species can often lead to the development of various chronic diseases. Genome sequencing of several members of the genus Chlamydia revealed that there are only a few C. trachomatis genes with no homologs in the genome of C. pneumoniae, whilst C. pneumoniae contains ~200 genes not found in C. trachomatis. It is highly likely that those genes, unique to C. pneumoniae, significantly contribute to the particular tissue tropism of this organism. In this grant we propose to utilize C. trachomatis, L2 as a surrogate microorganism together with recently developed approaches for genetic manipulation of this chlamydial species in investigating unique host-cell interactions of three C. pneumoniae-specific inclusion membrane proteins (Incs). Initially, we would focus on CP0641, CP0401, and CP0707 putative Incs because our preliminary data demonstrated that a domain(s) of each proposed Inc is exposed to the eukaryotic host cytoplasm. Due to the unavailability of reliable genetic manipulation tools for generating mutations in C. pneumonaie, employing C. trachomatis, L2 as a surrogate microorganism may prove useful for studying not only C. pneumonaie-specific genes but also genes from other chlamydial species.